As detailed in the preliminary data, we have demonstrated that mishandling of dopamine via reduced VMAT2 expression causes a progressive loss of dopamine terminals and cell bodies in the substantia nigra, synuclein aggregation, and L-DOPA responsive behavioral deficits, making the VMAT2 deficient mice a unique progressive model of PD. WE have shown that several environmental toxicants alter VMAT2 function. We hypothesize that the reduced ability to store dopamine causes oxidative stress in the presynaptic dopamine neuron, damage to cellular constituents, and the death of the neuron. Thus, the sxidative damage that occurs following improper storage of dopamine may underlie the disease process. We have recently shown that the redox state (dynamic balance between reduced and oxidized components) of neurons can be spatially resolved by subcellular compartment in that compounds used in animal models of Parkinson's disease can preferentially oxidize cytoplasmic, mitochondria!, or nuclear redox components, such as thioredoxin (Trx) or glutathione (GSH). Thus, the perturbation of redox state by altered dopamine homeostasis may be a key mediator of toxicity. Given the critical role of VMAT2, a-synuclein, and redox state the dopamine system, the goal of this project is to identify the interactions among these three components in order to better understand the underlying pathogenic processes that mediate cellular damage n Parkinson's disease. Hypothesis: Altered compartmentalization of dopamine disrupts the redox balance of the neuron resulting in enhanced vulnerability of the dopamine neuron Aim 1. To determine the role of asynuclein in regulation of the localization and function of VMAT2. We have preliminary evidence of a direct regulatory interaction of VMAT2 and a-synuclein. In this aim we will determine the nature of this molecular and functional interaction using site-directed mutagenesis, confocal microscopy, and vesicular uptake assays. Aim 2. t To determine if a-synuclein expression alters the vulnerability of dopamine-mediated injury due to reduced VMAT2 expression. By harvesting midbrain dopamine neurons from animals with differential expression of VMAT2 and a-synuclein, we will examine the effects of increased cytoplasmic dopamine and a-synuclein on dopamine-mediated cell death. Aim 3. To determine the effects of reduced or increased expression of VMAT2 on vulnerability to exogenous dopamine or MPTP. Aim 4: To expand our current kinetic-dynamic model of dopamine metabolism to encompass targets of environmental toxicants. We will extend our model to account for the oxidative stress that occurs with mitochondria! dysfunction and the impact of a-synculein on key components of the dopamine system. Completion of the above specific aims will improve our mechanistic understanding of how environmental and genetic factors disrupt proper storage of dopamine and contribute to the pathogenesis of Parkinson's disease